Myosin V is the best characterized vesicle transporter in vertebrates, but it has been unknown as to whether all members of the myosin V family share a common, evolutionarily conserved mechanism of action. Here we show that myosin V from Drosophila has a strikingly different motor mechanism from that of vertebrate myosin Va and it is a non-processive, ensemble motor. Our steady-state and transient kinetic measurements on single-headed constructs reveal that a single Drosophila myosin V molecule spends most of its mechanochemical cycle time detached from actin, therefore, it has to function in processive units that comprise several molecules. Accordingly, in in vitro motility assays, double-headed Drosophila myosin V requires high surface concentrations in order to exhibit a continuous translocation of actin filaments. Our comparison between vertebrate and fly myosin Vs demonstrates that the well-preserved function of myosin V motors in cytoplasmic transport can be accomplished by markedly different underlying mechanisms. We are also expressing GFP-tagged full-length myosin V and fragments of myosin V in Drosophila S2 cells and imaging the dynamics of myosin-associated vesicles in order to understand its role in membrane trafficking.